Limits...
Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.

Cao H, Dai P, Wang W, Li H, Yuan J, Wang F, Fang CM, Pitha PM, Liu J, Condit RC, McFadden G, Merghoub T, Houghton AN, Young JW, Shuman S, Deng L - PLoS ONE (2012)

Bottom Line: Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1.These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway.Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists.

View Article: PubMed Central - PubMed

Affiliation: Dermatology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

Show MeSH

Related in: MedlinePlus

Heat-VAC induced production of type I IFN is dependent on IRF7 and IFNAR1.Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from IRF7−/− (A), IFNAR1−/− (B) mice and age-matched WT controls. pDCs (2×105) were stimulated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351467&req=5

pone.0036823-g007: Heat-VAC induced production of type I IFN is dependent on IRF7 and IFNAR1.Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from IRF7−/− (A), IFNAR1−/− (B) mice and age-matched WT controls. pDCs (2×105) were stimulated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown.

Mentions: Transcription factor IRF7 is critical for type I IFN induction in pDCs and it plays essential role for host antiviral immunity 34. We have previously reported that IRF7 is important for type I IFN induction by myxoma virus in pDCs 15. Here we show that Heat-VAC-induced IFN-α/β production also requires IRF7 (Fig. 7A). Similar to what we observed for myxoma virus, Heat-VAC induction of type I IFN in pDCs requires IFNAR1, which mediates the type I IFN positive feedback loop (Fig. 7B).


Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.

Cao H, Dai P, Wang W, Li H, Yuan J, Wang F, Fang CM, Pitha PM, Liu J, Condit RC, McFadden G, Merghoub T, Houghton AN, Young JW, Shuman S, Deng L - PLoS ONE (2012)

Heat-VAC induced production of type I IFN is dependent on IRF7 and IFNAR1.Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from IRF7−/− (A), IFNAR1−/− (B) mice and age-matched WT controls. pDCs (2×105) were stimulated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351467&req=5

pone.0036823-g007: Heat-VAC induced production of type I IFN is dependent on IRF7 and IFNAR1.Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from IRF7−/− (A), IFNAR1−/− (B) mice and age-matched WT controls. pDCs (2×105) were stimulated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown.
Mentions: Transcription factor IRF7 is critical for type I IFN induction in pDCs and it plays essential role for host antiviral immunity 34. We have previously reported that IRF7 is important for type I IFN induction by myxoma virus in pDCs 15. Here we show that Heat-VAC-induced IFN-α/β production also requires IRF7 (Fig. 7A). Similar to what we observed for myxoma virus, Heat-VAC induction of type I IFN in pDCs requires IFNAR1, which mediates the type I IFN positive feedback loop (Fig. 7B).

Bottom Line: Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1.These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway.Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists.

View Article: PubMed Central - PubMed

Affiliation: Dermatology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

Show MeSH
Related in: MedlinePlus